Bottom Line:
Treatment with both flavonoids caused significant reduction in the mRNA level of COX-2, but the anti-inflammatory effect of the methylated analogue was more effective than the unmethylated one.Furthermore, both flavonoids reduced significantly the level of extracellular H2O2 compared to the control cells.In conclusion, the methylated apigenin analogue could avoid LPS-induced intestinal inflammation and it could be applied in the future as an effective anti-inflammatory compound.

ABSTRACTThe in vitro anti-inflammatory effect of apigenin and its trimethylated analogue (apigenin-trimethylether) has been investigated in order to evaluate whether these flavonoids could attenuate LPS-induced inflammation in IPEC-J2 non-transformed intestinal epithelial cells. Levels of IL-6, IL-8, TNF-α, and COX-2 mRNA were measured as a marker of inflammatory response. The extracellular H2O2 level in IPEC-J2 cells was also monitored by Amplex Red assay. Our data revealed that both compounds had significant lowering effect on the inflammatory response. Apigenin (at 25 μM) significantly decreased gene expression of IL-6 in LPS-treated cells, while apigenin-trimethylether in the same concentration did not influence IL-6 mRNA level. Both apigenin and apigenin-trimethylether reduced IL-8 gene expression significantly. TNF-α mRNA level was decreased by apigenin-trimethylether, which was not influenced by apigenin. Treatment with both flavonoids caused significant reduction in the mRNA level of COX-2, but the anti-inflammatory effect of the methylated analogue was more effective than the unmethylated one. Furthermore, both flavonoids reduced significantly the level of extracellular H2O2 compared to the control cells. In conclusion, the methylated apigenin analogue could avoid LPS-induced intestinal inflammation and it could be applied in the future as an effective anti-inflammatory compound.

fig2: Rate of living IPEC-J2 cells after apigenin and apigenin-trimethylether treatment. Cell viability was tested using Neutral Red uptake method. Effect of flavones on cell viability was studied in a dose- (a) and time-dependent (b) manner. Data are shown as means + standard deviations.

Mentions:
Viability of IPEC-J2 cells was monitored after apigenin and apigenin-trimethyether treatment, respectively (see Figures 2(a) and 2(b)). Neutral Red uptake assay showed that there was no significant difference in number of viable IPEC-J2 cells incubated with plain DMEM containing 0.1% DMSO. At 25 μM treatment dose in case of both flavones for 1 h did not reduce the number of viable enterocytes significantly. More than 44% of the IPEC-J2 cells were killed by 1 h treatment when apigenin-trimethylether was applied at 50 μM concentrations, while incubation with 100 μM destroyed 55.9 ±8.53% of cells. After 1 h exposure of 50 μM and 100 μM apigenin, reduced viability of IPEC-J2 cells was detected (rate of living cells decreased to 80.9 ± 7.49% and 64.1 ± 8.53%, resp., compared to the control samples). On the basis of the abovementioned data, it seemed to be safe to use both flavones in 25 μM concentration for further experiments. Viability was tested also on a time-dependent manner, using 25 μM concentrations of polyphenols. Living cell rate decreased to 75.3 ± 9.9% after 2 h apigenin-trimethylether exposure, while 4 h and 24 h treatment greatly reduced number of living IPEC-J2 cells (rate of viable cells was 70.3 ± 20.1 and 43.6 ± 18.3, resp.). Reduced viability was observed when enterocytes were treated with 25 μM apigenin for 4 h and 24 h, respectively (in preliminary studies rate of viable cells was 54.7 ± 3.84 and 45.0 ± 10.5, resp.). Therefore, 1 h treatment period was used in further experiments.

fig2: Rate of living IPEC-J2 cells after apigenin and apigenin-trimethylether treatment. Cell viability was tested using Neutral Red uptake method. Effect of flavones on cell viability was studied in a dose- (a) and time-dependent (b) manner. Data are shown as means + standard deviations.

Mentions:
Viability of IPEC-J2 cells was monitored after apigenin and apigenin-trimethyether treatment, respectively (see Figures 2(a) and 2(b)). Neutral Red uptake assay showed that there was no significant difference in number of viable IPEC-J2 cells incubated with plain DMEM containing 0.1% DMSO. At 25 μM treatment dose in case of both flavones for 1 h did not reduce the number of viable enterocytes significantly. More than 44% of the IPEC-J2 cells were killed by 1 h treatment when apigenin-trimethylether was applied at 50 μM concentrations, while incubation with 100 μM destroyed 55.9 ±8.53% of cells. After 1 h exposure of 50 μM and 100 μM apigenin, reduced viability of IPEC-J2 cells was detected (rate of living cells decreased to 80.9 ± 7.49% and 64.1 ± 8.53%, resp., compared to the control samples). On the basis of the abovementioned data, it seemed to be safe to use both flavones in 25 μM concentration for further experiments. Viability was tested also on a time-dependent manner, using 25 μM concentrations of polyphenols. Living cell rate decreased to 75.3 ± 9.9% after 2 h apigenin-trimethylether exposure, while 4 h and 24 h treatment greatly reduced number of living IPEC-J2 cells (rate of viable cells was 70.3 ± 20.1 and 43.6 ± 18.3, resp.). Reduced viability was observed when enterocytes were treated with 25 μM apigenin for 4 h and 24 h, respectively (in preliminary studies rate of viable cells was 54.7 ± 3.84 and 45.0 ± 10.5, resp.). Therefore, 1 h treatment period was used in further experiments.

Bottom Line:
Treatment with both flavonoids caused significant reduction in the mRNA level of COX-2, but the anti-inflammatory effect of the methylated analogue was more effective than the unmethylated one.Furthermore, both flavonoids reduced significantly the level of extracellular H2O2 compared to the control cells.In conclusion, the methylated apigenin analogue could avoid LPS-induced intestinal inflammation and it could be applied in the future as an effective anti-inflammatory compound.

ABSTRACTThe in vitro anti-inflammatory effect of apigenin and its trimethylated analogue (apigenin-trimethylether) has been investigated in order to evaluate whether these flavonoids could attenuate LPS-induced inflammation in IPEC-J2 non-transformed intestinal epithelial cells. Levels of IL-6, IL-8, TNF-α, and COX-2 mRNA were measured as a marker of inflammatory response. The extracellular H2O2 level in IPEC-J2 cells was also monitored by Amplex Red assay. Our data revealed that both compounds had significant lowering effect on the inflammatory response. Apigenin (at 25 μM) significantly decreased gene expression of IL-6 in LPS-treated cells, while apigenin-trimethylether in the same concentration did not influence IL-6 mRNA level. Both apigenin and apigenin-trimethylether reduced IL-8 gene expression significantly. TNF-α mRNA level was decreased by apigenin-trimethylether, which was not influenced by apigenin. Treatment with both flavonoids caused significant reduction in the mRNA level of COX-2, but the anti-inflammatory effect of the methylated analogue was more effective than the unmethylated one. Furthermore, both flavonoids reduced significantly the level of extracellular H2O2 compared to the control cells. In conclusion, the methylated apigenin analogue could avoid LPS-induced intestinal inflammation and it could be applied in the future as an effective anti-inflammatory compound.